Exploration of Simiao-Yongan Decoction on knee osteoarthritis based on network pharmacology and molecular docking

Use network pharmacology combined with molecular docking to study the effects of Simiao-Yongan Decoction (SMYAD) intervenes in Knee Osteoarthritis (KOA) related targets and signaling pathways, and explores the molecular mechanism of SMYAD in treating KOA. The active ingredients and targets of SMYAD, which concluded 4 traditional Chinese medicines, were screened in TCMSP, and the related gene targets of KOA were screened in the disease databases GeneCards, MalaCards, DisGeNET, and Comparative Toxicogenomics Database, and their intersection data were obtained after integration. And used Cytoscape 3.9.1, the software topologies the network diagram of “compound—drug-active ingredient-target protein-disease.” Obtains the protein-protein interaction network diagram through STRING, and enriches and analyzes the obtained core targets. Carry out molecular docking matching verification on the main active ingredients and key targets of the drug. 106 active ingredients and 175 targets were screened from SMYAD to intervene in KOA, 36 core targets were obtained through protein-protein interaction screening, and 10 key targets played an important role. The enrichment results showed that the biological process of gene ontology mainly involved positive regulation of gene expression, negative regulation of apoptosis process, and positive regulation of apoptosis process. KEGG signaling pathway mainly involves AGE-RAGE signaling pathway in diabetic complications, TNF signaling pathway, hypoxia-inducible factor-1 signaling pathway, IL-17 signaling pathway. The pathway of Reactome mainly involves interleukin-4 and interleukin-13 signaling, cytokine signaling in immune system, immune system, apoptosis. Molecular docking showed that the mainly effective components of SMYAD can fully combine with TNF, IL1B, IL6, and CASP3. The results show that the main active ingredients and potential mechanism of action of SMYAD in the treatment of KOA have the characteristics of multiple targets and multiple pathways, which provides ideas and basis for further in-depth exploration of its specific mechanism.


Introduction
Knee osteoarthritis (KOA) is the most common joint inflammatory disease in orthopedics.The main feature is the gradual degeneration and loss of articular cartilage, accompanied by changes in joint structure and function. [1]After entering the 21st century, because of the increase in life expectancy and increase in body mass index due to the increase of population of old people, the prevalence of KOA has increased significantly and tended to be younger. [2]Currently, KOA accounts for 85% of the global burden of osteoarthritis, and the incidence of symptomatic KOA is 8.1%. [3]The main clinical manifestations of KOA are inconvenient movement of the limbs, difficulty in moving, and pain and swelling around the joints, which are more obvious after waking up in the morning, and can lead to paralysis in severe cases in the later stage. [2][6] Therefore, it is crucial to understand the pathogenic process of KOA and the cellular and molecular mechanisms involved.The current process of osteoarthritis of the knee contains 2 basic aspects: the action of different joint elements and posttraumatic osteoarthritis formation. [7,8]On one hand, in nontraumatic circumstances, the development of knee osteoarthritis is a degenerative pathology associated with aging.Injuries to the components of the knee joint, such as synovitis, ligament, meniscus, free bodies, popliteal cysts, and chondromalacia of the patella, can also result in the development of knee osteoarthritis.In addition, knee osteoarthritis can be brought on by putting on too much weight, adopting bad gait patterns, squatting for extended periods of time, and exposing the knee joint to the cold. [4,9]On the other hand, posttraumatic osteoarthritis development, a progressive form of arthritis in the knee following trauma, is a significant contributor to osteoarthritis of the knee.It has clinical symptoms with osteoarthritis, but it also has a clear history of trauma, such as transarticular fractures. [10,11]At present, the most common manifestation of KOA is pain, and the effective drugs for pain relief are nonsteroidal antiinflammatory drugs. [12]However, such drugs are only suitable for short-term pain relief, and cannot control the wear and tear of articular cartilage, and cannot solve the fundamental problem. [13]Moreover, such drugs may cause gastrointestinal discomfort such as nausea and vomiting, and increase the risk of gastrointestinal bleeding. [14]Therefore, it is imperative to develop a drug that can maintain the curative effect of the drug and reduce side effects as much as possible.
Traditional Chinese medicine (TCM) attributes KOA to the categories of "Bi Syndrome" and "Bone Bi." TCM has a long history of treating KOA, and the basic theoretical system of "arthromyopathy" was established as early as in the "Huangdi Neijing."TCM has the advantages of definite curative effect and high safety in the treatment of KOA.It has a good clinical effect on the treatment of KOA and plays a vital role in the diagnosis and treatment of KOA.Many doctors in modern times use Simiao-Yongan Decoction (SMYAD) to treat arthritis.The results of modern pharmacological research also show that SMYAD has antiinflammatory, analgesic, and microcirculation  improvement functions, [15] and there have been clinical research reports, SMYAD has a definite curative effect on gouty arthritis, but there is no relevant research on the active substances and specific molecular mechanisms of SMYAD. [16]etwork pharmacology is a new discipline formed by the fusion of pharmacology with multidisciplinary methods, which can comprehensively reflect the molecular mechanism of action of drugs on disease networks. [17]In this study, using the methods of network pharmacology and molecular docking, starting from the chemical substance basis of SMYAD, the important targets and potential molecular mechanisms of drug regulation on KOA are more systematically and deeply explored, to better serve the treatment of KOA.It provides a theoretical reference for the clinical treatment of KOA and provides new ideas for the development of new drugs for the treatment of KOA. [18] Materials and methods

Screen the active ingredients of SMYAD and the protein targets of KOA
Jin Yinhua (or honeysuckle), Xuan Shen (or Genuine Ginseng), Dang Gui (or angelica), and Gan Cao (or licorice) were collected through TCMSP (http://tcmspw.com/tcmsp.php)database.Taking the oral bioavailability ≥30% and drug similarity (DL) ≥0.18 as the standard, the effective active ingredients of the drug were screened, and the targets corresponding to the active ingredients were queried, [19] targets to obtain effective targets.In GeneCards (https://www.genecards.org/),MalaCards (https://www.malacards.org/),DisGeNet database (https:// www.disgenet.org/),and Comparative Toxicogenomics Database (http://ctdbase.org/,last update by June) obtain KOArelated targets. [20,21]The target of the obtained compound was intersected with the KOA target, and the visual connection of "compound-drug-active ingredient-target protein-disease" was constructed. [22]The raw data used in our investigation can be replicated and were taken from the relevant databases.Our tests' outcomes are essentially unaffected by any disparities in the data, despite the fact that the databases are updated dynamically.

Construction of protein-protein interaction network
Protein-protein interactions underlie most BPs in living cells and are critical for understanding cell physiology in both normal and disease states.In this study, the protein-protein interaction (PPI) network analysis was performed on the obtained intersection gene sets using the string database (http://string-db.org/), and the species was restricted to "Homo sapiens" with a confidence value >0.4. [23]The PPI network was constructed by Cytoscape software (version 3.9.1).In addition, using the plug-in CytoHubba algorithm of Cytoscape software, the targets with a maximum degree greater than or equal to half of the maximum degree were selected as core targets, and the top 10 targets in descending order of degree were selected as key targets.

GO, KEGG, and Reactome enrichment analysis
Import the obtained intersection targets into the HiPlot database (https://hiplot.com.cn/) and Reactome database (https://reactome.org/), [24,25] and conduct pathways with humans as the research object enrichment analysis.Among them, Gene Ontology (GO) analysis includes 3 categories: biological process (BP), cellular component (CC), and molecular function, and according to the P. adjust.The top 20 enriched pathways of Reactome were selected according to the P. adjust.To select the top 20 annotated by KEGG, according to P value.The results are displayed in column charts, bubble charts, and network correlation diagrams.

Molecular docking analysis
The 3D structure of the proposed docking target in mol2 format from the Pubchem database, open the small ligand molecule with AutodockTools 1.5.6,perform hydrogenation, charging, detection of the ligand root, search and define rotatable bonds, and then save it as a pdbqt file.Download the core 3-dimensional structure of the target protein as a docking protein from the RCSB protein database (www.rcsb.org/).Add all hydrogen atoms in AutodockTools 1.5.6 to open, calculate the Gasteiger charge, combine nonpolar hydrogen, define it as an acceptor, and save it as a pdbqt file.Determine the coordinates and box size of the Vina molecular docking, set the exhaustiveness of the parameters to 15, and take the default values for other parameters.Autodockvina 1.1.2was used for semiflexible docking, and the conformation with the best affinity was selected as the final docking conformation.

Active ingredient and target protein analysis of SMYAD
Total of 126 active ingredients were retrieved through the TCMSP database, including 2 angelica, 23 honeysuckle, 9 Genuine Ginseng, and 92 licorice.After deleting repeated ingredients, a total of 120 chemical ingredients were obtained.There are a total of 2042 corresponding targets.The protein names were annotated according to the standard and the duplicate targets were deleted through the Uniprot database.A total of 202 targets were screened (Tables S1, Supplemental Digital Content, http://links.lww.com/MD/K40 and S2, http://links.lww.com/MD/K42).The keyword "Knee Osteoarthritis" was searched in the disease database, and the corresponding targets were obtained after screening, and the union was taken, and the KOA-related targets were obtained after deleting duplicate items (Table S3, Supplemental Digital Content, http://links.lww.com/MD/K43, Figure 1A).The targets corresponding to the active ingredients obtained through screening were intersected with KOA-related targets to obtain 175 intersection targets (Figure 1B).Use Cytoscape software to build a "compound-drug-active ingredient-target protein-disease" network (Figure 2).Using Cytoscape to calculate the weight of active ingredients, the top 8 active ingredients in degree value are quercetin, luteolin, kaempferol, beta-sitosterol, naringenin, 7-methoxy-2-methyl isoflavone, formononetin, licochalcone a.These may be the key active ingredients of SMYAD for the treatment of KOA (Table 1).

Construction of protein-protein interaction network
Import the intersection targets into the String database for PPI network.According to the CytoHubba algorithm, 36 core targets and the top 10 key targets of degree are obtained (Figure 3).The active ingredients corresponding to the core targets were intersected with the previously obtained 8 active ingredients that may be SMYAD for the treatment of KOA.The results showed that the intersection overlapped with the previous 8 active ingredients, so it was speculated that quercetin, luteolin, kaempferol, beta-sitosterol, naringenin, 7-methoxy-2-methyl isoflavone, formononetin, licochalcone a may be the key active ingredients of SMYAD in the treatment of KOA.And quercetin, luteolin, kaempferol, and beta-sitosterol contain the largest number of intersection targets, so they are considered to be the main effector substances.

Gene function and pathway enrichment results
Through the enrichment analysis of 36 core targets, a total of 536 GO annotations were obtained.According to the value of P. adjust, the top 10 BP, CC, and molecular function of GO annotations were screened out (Table S4, Supplemental Digital Content, http://links.lww.com/MD/K44).Among them, BP mainly involves positive regulation of gene expression, positive regulation of transcription, DNA-templated, positive regulation of pri-miRNA transcription from RNA polymerase II promoter, response to drug, negative regulation of apoptotic process, positive regulation of apoptotic process, lipopolysaccharide-mediated signaling pathway, positive regulation of transcription from RNA polymerase II promoter, response to estradiol, positive regulation of smooth muscle cell proliferation.CC mainly involves macromolecular complex, extracellular space, Caveola, transcription factor complex, nucleoplasm, cytosol, cytoplasm, nucleus, mitochondrion, extracellular region.MM mainly includes enzyme binding, transcription factor binding,  3).Reactome was enriched to 228 pathways.According to the value of P. adjust, the top 20 pathways were screened out, mainly involving interleukin-4 and interleukin-13 signaling, signaling by interleukins, cytokine signaling in immune system, immune system, interleukin-10 signaling, apoptosis (Figure 4D, Table 4).According to the results of comprehensive enrichment analysis, the main effect pathways of SMYAD in treating KOA are AGE-RAGE signaling pathway in diabetic complications and TNF signaling pathway.We downloaded the relevant pathway diagrams from the KEGG database for further analysis and found that IL6, TNF, CASP3, and IL1B are the key regulatory targets of the 2 pathways, and they highly overlap with the top 10 key proteins we screened (Figure 5).In addition, the 2 signaling pathways ultimately affect the expression of apoptotic signals, and these results are consistent with the enrichment analysis results of GO and Reactome (Figure 6).Therefore, we believe that based on the key proteins IL6, TNF, CASP3, and IL1B, the regulation of AGE-RAGE signaling pathway in diabetic complications and TNF signaling pathway to affect the occurrence of apoptosis is the key to the treatment of KOA by SMYAD.

Molecular docking
According to the results of enrichment analysis, we found that TNF, IL6, IL1B, and CAPSE3, the key proteins of SMYAD in the treatment of KOA, play an important role.Therefore, we carried out molecular docking of these 4 hub genes and important active compounds quercetin, luteolin, kaempferol, beta-sitosterol.Small molecule ligands can spontaneously bind to protein receptors when the binding energy is <0 kJ mol.If the binding energy is <−5.0 kJ mol or lower, it indicates that the 2 have better binding ability.Through the docking simulation, 16 docking results were generated (Table 5).Their binding energies are all <0 kJ mol, which means they all bind well.Finally, Pymol software was used to visualize the complexes of 4 groups of proteins and compounds with good docking effect (Figure 7).

Discussion
KOA is a kind of degenerative arthritis that mainly occurs in the elderly.It can be caused by a variety of causes, including articular cartilage damage, subchondral bone tissue, joint edge synovium, and joint tissue changes. [26]Clinically, in the face of patients with knee arthritis in the early and middle stages, nonsurgical treatment is often used to improve knee joint disorders, relieve pain, promote joint function recovery, and reduce articular cartilage damage. [27]Commonly used methods include active muscle strength training, activity adjustment, and changing activity patterns, and so on, and most patients can obtain relatively ideal therapeutic effects. [28]For patients with advanced knee arthritis because the articular cartilage is severely damaged and the bone is exposed and necrotic, extensive osteophytes, and subchondral cysts are prone to occur.Although there is no specific treatment at present, joint cavity washing can effectively relieve the pain, but the curative effect is not obvious. [29]In recent years, many experiments and clinical observations have confirmed that TCM has a good curative effect on KOA, [30] among which SMYAD has a good therapeutic effect on KOA.However, its active ingredients and mechanism are still unclear.Therefore, this study intends to discover the potential mechanism of SMYAD in the treatment of KOA through network pharmacology, and provide theoretical support for the application of TCM.One hundred twenty active ingredients of SMYAD were screened out from the TCMSP database.The top 8 active ingredients ranked according to degree value are quercetin, luteolin, kaempferol, beta-sitosterol, naringenin, 7-methoxy-2-methyl isoflavone, formononetin, licochalcone a.Therefore, it is speculated that these may be the key active ingredients of SMYAD in the treatment of KOA.Quercetin is a polyhydroxy flavonoid compound, which has biological activities such as antivirus, antitumor, and liver protection. [31]Related studies have found that quercetin can up-regulate SOD and TIMP-1, down-regulate MMP-13, and improve the degeneration of KOA. [32]Luteolin is a natural flavonoid found in many plants.It has a variety of pharmacological activities, such as antiinflammatory, antiallergic, and uric acid-lowering. [33]Studies have shown that luteolin can regulate the gene expression, secretion, and activity of MMP-3 by acting directly on articular chondrocytes, and play a potential chondroprotective role. [34]Kaempferol is also a kind of natural flavonoids, mainly found in tea, broccoli, apple, and other plants.A large number of clinical studies have shown that kaempferol and some kaempferol glycosides have a wide range of pharmacological activities, including antioxidation, antiinflammation, antidiabetes, antiosteoporosis and other effects. [35]tudies have found that kaempferol potentially improves the efficiency of arthritis cell therapy. [36]The above studies are consistent with the results of this study, suggesting that quercetin, luteolin, kaempferol, and other active ingredients may play a key role in the treatment of OP by SMYAD.According to Degree analysis of PPI network, the results showed that 10 core targets AKT1, TP53, TNF, IL6, VEGF-A, CASP3, IL1B, CTNNB1, ESR1, EGFR may play a key role in the treatment of KOA by SMYAD.VEGF is a well-known angiogenic factor.Related studies have shown that the expression of VEGF-A is a special feature of arthritis, and found that VEGF-A and VEGFR2 are related to the vascular invasion of cartilage. [37]Elevated VEGF-A levels are associated with the progression of OA.It is involved in the specific pathological processes of OA, including cartilage degeneration, osteophyte formation, subchondral bone cysts and sclerosis, synovitis and pain.Inhibition of VEGF-A signaling can reduce the progression of OA. [38] The results of GO functional enrichment analysis suggested that SMYAD treatment of KOA mainly involves positive regulation of gene expression, negative regulation of apoptosis process, and positive regulation of apoptosis process.KEGG signaling pathway mainly involves AGE-RAGE signaling pathway in diabetic complications, TNF signaling pathway, HIF-1 signaling pathway, IL-17 signaling pathway.The pathway of Recactome mainly involves interleukin-4 and interleukin-13 signaling, cytokine signaling in immune system, immune system, apoptosis.The biological activity of AGEs is considered to be mediated by age-specific receptors (RAGE), and the activation of RAGE is involved in key signaling pathways related to KOA. [39] On the one hand, AGEs induce inflammation by activating NF-κB and MAPK in various cell types including chondrocytes.On the other hand, AGEs may increase the levels of PGE and NO through the MAPK pathway to enhance the inflammatory response of OA chondrocytes.
In addition, AGEs stimulated endoplasmic reticulum stress in human chondrocytes through RAGE, leading to phosphorylation of eIF2α and p38 MAP kinases and activation of NF-κB, which in turn increased COX-2 expression and PGE2 production. [40]IL-17 is one of many inflammatory cytokines, mainly produced by T cells.The cells affected by IL-17 in joints are mainly chondrocytes.Imaging studies have shown that images of OA lesions are positively correlated with elevated levels of IL-17 in serum and synovial fluid.In addition, IL-17 can induce chondrocytes and synovial fibroblasts to release chemokines and induce chondrocytes to synthesize IL-1, thereby promoting the pathological progression of OA. [41] Hypoxia-inducible factor (HIF) is a transcription factor and a key regulator of cellular responses to hypoxia. [42]HIF-1 is an important factor in maintaining chondrocyte homeostasis and allowing cell differentiation.HIF-1 is a heterodimer composed of 2 subunits: HIF-1α and HIF-1.A study showed that HIF-1α plays an important role in the prevention of OA.HIF-1α downregulates MMP-13 by inhibiting β-catenin transcriptional activity in chondrocytes, thereby alleviating OA development.Lack of HIF-1α exacerbates cartilage catabolism.In addition, HIF-1 is also involved in the process of KOA synovial fibrosis. [43]Molecular docking showed that the important active compounds quercetin, luteolin, kaempferol, and beta-sitosterol could fully combine with key proteins TNF, IL6, IL1B, and CAPSE3.This may be the key molecular mechanism of SMYAD in treating KOA.
Even while our research has clearly shown the beneficial efficacy and mechanism of SMYAD in the treatment of KOA, our medication is safer for clinical use when compared to the side effects of conventional NSAIDs, such as peptic ulcers.Nonetheless, there are Figure 6.Reactome analysis.The diagrams show the human systems and locations of cellular responses involved in the key targets' actions, and so on.The 36 key targets focus on the immune system, among these, the mechanisms involved in the immune system make it a priority for us to understand and study in depth.

Table 5
The binding energy of molecular docking (kJ mol).still certain issues that require First of all, SMYAD is a complex formula with a wide range of effects from each of its constituent parts.Therefore, a review of the drug's plasmapheresis analysis is necessary to further elucidate the effects of each component.The few active chemicals we detected may not be sufficient.Second, until date, we have only verified theoretical partial molecular docking in a thorough and scientific manner.We plan to enhance the associated experiments in the future.

Conclusion
In summary, this study preliminarily verified and predicted the molecular mechanism of SMYAD in the treatment of KOA, and found that the active ingredients and mechanism of SMYAD in the treatment of KOA present the characteristics of multi-component, multi-target, and multi -pathway.

Figure 4 .
Figure 4. Results of the enrichment analysis graph.(A) KEGG enrichment analysis results graph, (B) signaling pathway and key target association maps based on KEGG enrichment analysis, (C) Barplot of the top 10 GO enrichment items, the GO enrichment items (BP, CC, MF) are arranged from up to low according to the adjusted P value, (D) bubble chart of Reactome enrichment analysis results.BP = biological processes, KEGG = Kyoto Encyclopedia of genes and genomes, CC = cell composition, GO = gene ontology, MF = molecular function.

Table 1
Screening of active ingredients of Simiao-Yongan Decoction from TCMSP database.

Table 2
The top 10 gene ontology enrichment items.

Table 3
The top 20 KEGG enrichment items.

Table 4
The top 10 Reactome enrichment items.